Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.
Whole-exome sequencing (WES) has emerged as the preferred method to identify disease genes for Mendelian disorders. Indeed, WES is proving particularly valuable for the diagnostic evaluation of individuals with phenotypically and genetically heterogeneous conditions such as suspected mitochondrial disease (McCormick et al. (2013) Neurotherapeutics 10:251-61). Mitochondrial diseases have a wide range of presenting disease manifestations, typically poor genotype-phenotype correlation of any one gene, and a wide range of phenotypically similar non-mitochondrial diseases that must be considered in the differential diagnosis for any given patient (Haas et al. (2007) Pediatrics 120:1326-1333). Known pathogenic mutations causing mitochondrial disease have already been identified in more than 150 nuclear genes and all 37 mtDNA genes (Calvo et al. (2010) Annu. Rev. Genomics Hum. Genet., 11:25-44), although most genes have been linked to only a small number of disease cases and mutations in these known genes collectively account for less than half of cases with suspected mitochondrial disease (Calvo et al. (2012) Sci. Transl. Med., 4:118ra110). Additional pathogenic candidates abound as there are up to 1,500 mitochondrial proteins that are largely nuclear-encoded, of which the MitoCarta set of 1,034 proteins has undergone robust experimental validation and accounts for approximately 85% of all mitochondrial proteins (Pagliarini et al. (2008) Cell 134:112-123). The MitoCarta set includes many known disease genes, including all but 4 nuclear genes (TAZ, PUS1, RRM2B, TYMP) of 77 (Calvo et al. (2012) Sci. Transl. Med., 4:118ra110) previously linked to mitochondrial respiratory chain disease (Tucker et al. (2010) Curr. Neurol. Neurosci. Rep., 10:277-285) and 80 of the nuclear genes on the 101 gene sequencing panel for mitochondrial disease and related disorders that is currently available in the clinical diagnostic setting at GeneDx (Gaithersburg, Md.). Targeted sequence analysis of the MitoCarta gene set together with the mtDNA genome has been estimated to be likely to identify pathogenic causes in at least 47% of all individuals with suspected primary mitochondrial disease (Calvo et al. (2012) Sci. Transl. Med., 4:118ra110). Therefore, sequence analysis of the MitoCarta nuclear gene set, the mtDNA genome, and the entire nuclear exome can reasonably be expected to facilitate genetic diagnosis in more than half of all patients with suspected mitochondrial disease, while also presenting the simultaneous opportunity for novel disease gene discovery. Such analysis is now technically feasible by application of massively parallel sequencing methodologies that have emerged in both the research and clinical settings.
A single unified platform has not been available to reliably permit simultaneous interrogation of all known and potential causes of suspected mitochondrial disease and phenotypically overlapping disorders. Exome capture kits are not all equally designed, do not capture the same target regions, and do not all perform with the same efficiency. Indeed, the early versions of commercially available whole-exome capture kits were found to target significantly different genomic regions and to vary greatly in their overall performance (Asan et al. (2011) Genome Biol., 12:R95; Kiialainen et al. (2011) PLoS One 6:e16486). In addition, no whole-exome capture kit has been optimized to provide highly reliable capture of the MitoCarta nuclear gene set and to provide targeted capture of the mtDNA genome. While off-target capture of the mtDNA genome is inevitable in any whole-exome capture kit, this is typically highly non-reproducible with insufficient coverage to either provide reliable interrogation of the complete mtDNA genome sequence or sensitively detect heteroplasmic mtDNA mutations.